Epigenetic control of Transcription Factor activity
Final Report Abstract
Successful development of complex organisms relies on accurate execution of transcriptional programs. Binding of transcription factors (TFs) to cis-regulatory elements (CREs) translates genetic information into appropriate gene expression patterns. In Eukaryotes, DNA is wrapped around histones representing a physical barrier preventing the spurious binding of TFs. The compaction level of chromatin is tightly regulated by multiple processes including post-translational modification of histones and DNA. Therefore, activity of a CREs is defined by the interpretation of its genetic information by TFs as well as the epigenetic signals present in its chromatin environment. It is commonly assumed that interactions between these two regulatory layers is a key to precisely control complex gene expression patterns required for development. However, the mechanisms by which DNA and chromatin modifications influence the binding of TFs are largely unknown. Enhancers are cis-regulatory elements that control the expression of genes involved in the acquisition of cellular identity during development and its maintenance in healthy tissues. DNA methylation is a repressive epigenetic mark present at over 80% of the cytosines in the mammalian genome. At active enhancers DNA methylation is reduced down to 10-60%. This incomplete demethylation implies the existence of epigenetic heterogeneity at active enhancers in a homogeneous population of cells. Presence of DNA methylation at enhancers could inhibit transcription factor (TF) binding and reduce their activity within the fraction of cells where it occurs. Alternatively, since the binding of certain TFs is insensitive to DNA methylation, residual methylation could be neutral to enhancer activity. To tease these two scenarios apart, we performed Single Molecule Footprinting in mouse embryonic stem cells to simultaneously quantify DNA methylation, chromatin accessibility and TF binding on individual DNA molecules genomewide. Using this data, we tested if chromatin accessibility and TF binding can occur at enhancers when their target DNA molecules are methylated. For most enhancers, chromatin accessibility occurs at similar levels on methylated and unmethylated molecules, suggesting that their activity is insensitive to the presence DNA methylation. However, we identified a subset of enhancers that showed reduced accessibility on methylated molecules, indicating a possible regulation of these loci by DNA methylation. This feature is not unique to embryonic stem cells as new putative methyl-sensitive enhancers can be identified in somatic cell-lines of the neuronal, myeloid and erythroid lineage. Genetic perturbation of DNA methylation leads to anti-correlated changes in chromatin accessibility and transcription factor binding at putative methyl-sensitive enhancers, suggesting a direct epigenetic control of TF binding at these loci. In summary, we identify enhancers that are regulated by DNA methylation in multiple cell-types and identify contexts of direct TF binding inhibition providing a mechanistic basis for their epigenetic regulation. This demonstrates that in addition to safeguarding the genome from spurious activation, DNA methylation functions through the modulation of transcription factor occupancy levels at enhancers. Together, the proposed work contributes to our understanding of how epigenetic modifications contribute to transcriptional control by modulating the access of TFs to their target CREs in the context of cell fate choices.
Publications
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Genome-wide quantification of transcription factor binding at single-DNA-molecule resolution using methyl-transferase footprinting. Nature Protocols, 16(12), 5673-5706.
Kleinendorst, Rozemarijn W. D.; Barzaghi, Guido; Smith, Mike L.; Zaugg, Judith B. & Krebs, Arnaud R.
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Molecular Co-occupancy Identifies Transcription Factor Binding Cooperativity In Vivo. Molecular Cell, 81(2), 255-267.e6.
Sönmezer, Can; Kleinendorst, Rozemarijn; Imanci, Dilek; Barzaghi, Guido; Villacorta, Laura; Schübeler, Dirk; Benes, Vladimir; Molina, Nacho & Krebs, Arnaud Regis
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Studying transcription factor function in the genome at molecular resolution. Trends in Genetics, 37(9), 798-806.
Krebs, Arnaud R.
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Single-molecule footprinting identifies context-dependent regulation of enhancers by DNA methylation. Molecular Cell, 83(5), 787-802.e9.
Kreibich, Elisa; Kleinendorst, Rozemarijn; Barzaghi, Guido; Kaspar, Sarah & Krebs, Arnaud R.
